Interleukin-18 (IL-18) is a potent cytokine that plays a role in both innate and acquired immune responses. IL-18 was first identified for its ability to induce the production of IFN-γ, one of the primary effector molecules of a T helper (Th) 1-type immune response (Okamura et al (1995) Nature 378:88-91). It was shown subsequently that, in certain settings, IL-18 can also contribute to the development of additional responses such as Th2 (Nakanishi et al (2001) Annu Rev Immunol. 19:423-74). In addition to its stimulatory effect on cluster of differentiation (CD) 4+ and CD8+ T cells, IL-18 has been shown to have a broad spectrum of inflammation-related effects on various cells types including neutrophils, monocytes/macrophages, natural killer (NK) cells, epithelial and endothelial cells.
IL-18 belongs to the interleukin-1 (IL-1) superfamily, and like IL-1 beta (IL-1β) it is first synthesised as an inactive intracellular polypeptide, pro-IL-18 (24 kDa). Enzymatic cleavage of the pro-peptide releases the biologically active mature form of IL-18 (18 kDa). IL-18 is produced by macrophages and other cells involved in the immune response. Apart from its physiological role, IL-18 also contributes to severe inflammatory reactions, providing indication of its role in certain inflammatory disorders such as autoimmune diseases. IL-18 is regulated in part by endogenous IL-18 binding protein (IL-18BP) which binds to IL-18 and blocks its binding to the IL-18 receptor (IL-18R), thereby quenching the IL-18 activity. However, in certain circumstances, this negative feedback loop is insufficient to adequately quench the biological effects of IL-18 whilst still providing enough IL-18 biological activity to initiate innate immune responses.
The biological activity of IL-18 is generated when IL-18 binds to cell-bound IL-18R, initiating cell signaling which leads to expression of other cytokines. The IL-18R is a heterodimer comprising two IgG like polypeptide chains that form a functional complex in which the β chain contains the IL-18 binding motif and the α chain contains an intracellular signalling domain (TIR) (Torigoe et al (1997) J Biol Chem 272:25737). IL-18 appears to first bind IL-18Rα and the β chain is then recruited to form the functional heterodimer. The binding of IL-18 to the α/β complex results in activation of the intracellular signalling cascade. Thus, blocking the binding of IL-18 to IL-18Rα may be useful in quenching unwanted IL-18.
IL-18 has been implicated in a number of human diseases, primarily autoimmune or inflammatory diseases and cancer. Psoriasis patients have increased IL-18 both in the skin lesions and in the circulation (Flisiak et al. (2006) 11:194). Elevated plasma IL-18 levels have been observed in cerebrospinal fluid and plasma of patients with multiple sclerosis (Fassbender et al. (1999) Neurology 53:1104; Nicoletti et al. (2001) Neurology 57:342). Patients with inflammatory bowel diseases (e.g. Crohn's disease, ulcerative colitis) have elevated IL-18 levels in the circulation (Ludwiczek et al (2005) Eur. Cytokine Netw. 16:27; Wiercinska-Drapalo et al (2005) World J Gastroenterol. 11:605). Patients with rheumatoid arthritis (RA) have elevated IL-18 levels in the synovial fluid (Gracie et al (1999) J Clin Invest 104:1393). IL-18 has also been shown to be increased in the serum of patients with other arthritic diseases with systemic manifestations such as Still's disease (Kawashima et al (2001) Arthritis Rheum. 44(3):550-60) and systemic juvenile idyopathic arthritis (sJIA) (Maeno et al (2002) Arthritis Rheum. 46(9):2539-41; Shimizu et al (2010) Rheumatology 49(9):1645-53) and has been proposed as a marker of disease severity (Kawagushi et al (2001) Arthritis Rheum. 44(7):1716-7; Lotito et al (2007) J Rheumatol. 34(4):823-30). In these autoinflammatory disorders, circulating IL-18 was shown to be further increased during the active phase of the disease and in a subset of patients developing complications such as macrophage activation syndrome (Shimizu et al (2010) Rheumatology 49(9):1645-53). IL-18 levels were also shown to be increased both systemically and in the pulmonary tissues of Chronic Obstructive Pulmonary Disease (COPD) patients, where IL-18 is associated with alveolar macrophages, CD8+ T cells and airway epithelial cells (Imaoka et al (2009) Eur Respir J. 31:287-97; Kang et al. (2007) J. Immunol. 178(3):1948-59; Petersen et al. (2007) Lung 185:161-71; Rovina et al (2009) Respir Med. 103:1056-62). IL-18 may also contribute to COPD co-morbidities (Petersen et al (2007) Lung 185:161; Larsen et al (2008) Cardiovasc Res 80:47).
In cardiovascular diseases, elevated plasma or serum levels of circulatory IL-18 were measured in acute coronary syndrome, myocardial infarction, coronary atherosclerosis, and unstable angina (Mallat et al (2002) Heart 88:467; Hulthe et al (2006) Atherosclerosis 188:450; Mallat et al (2001) Circulation 104:1598). Serum IL-18 levels are also associated with intima-media thickness of the carotid arteries (Yamagami et al (2004) Arterioscler Thromb Vasc Biol 25:1458).
Several types of cancers are also associated with elevated IL-18 expression levels. However the role of IL-18 may be dual in malignant diseases (Dinarello (2008) Cancer & Metastasis Rev 25:307). On one hand, IL-18 may be pro-angiogenic and thereby may promote tumor development, on the other hand it may stimulate cellular immune responses, e.g. NK cell mediated cytotoxicity and thereby act as an anti-tumorigenic agent (Kim et al. (2007) Oncogene 26:1468.).
Many of these clinical observations have also been corroborated in animal models of human disease. These studies have addressed both disease mechanisms dependent on IL-18 and IL-18 as a potential target for intervention. Development of CNS lesions in experimental autoimmune encephalitis (EAE), the most widely used animal model for multiple sclerosis, was shown to be dependent on IL-18Rα engagement (Gutcher et al. (2006) Nat Immunol 7:946.). The administration of IL-18 to animals was shown to enhance disease in the collagen induced arthritis model of RA (Gracie et al. (1999) J Clin Invest 104:1393), while lack of IL-18 or IL-18 neutralising therapies attenuated the severity of the disease (Banda et al. (2003) J Immunol 170:2100). IL-18 knockout mice are resistant to experimentally induced colitis (Hovet et al (2001) Gastroenterol 121:1372), whereas transgenic animals overexpressing IL-18 are more susceptible (Ishikura et al. (2003) J Gastroenterol Hepatol 18:960). IL-18 neutralising treatment attenuates intestinal inflammation (Lochner and Foster (2002) Pathobiol 70:164).
In an animal model developed for COPD, cigarette smoke was shown to increase IL-18 expression locally in the lung tissue (Kang et al (2008) Clin Invest 118:2771, Kang et al (2007) J Immunol 178:1948). In this model, impairment of IL-18 signaling via disruption of IL-18Rα gene resulted in a decreased lung inflammation, cell apoptosis and emphysema (Kang et al (2007) J Immunol 178:1948). Conversely, over-expression of IL-18 in the lung of mice was associated with pulmonary inflammation characterized by the recruitment of CD8+ T cells, macrophages, neutrophils and eosinophils as well as lung with an emphysematous phenotype (Hoshino et al (2007) Am J Respir Crit Care Med 176:49). In addition to chronic lung inflammation, IL-18 may be associated with virally or bacterially-triggered exacerbations. In a cigarette smoke mouse model, IL-18 levels were shown to be further enhanced by treatment of mice with poly(I:C) or infection with influenza and to contribute to the inflammation, alveolar remodeling and apoptosis (Kang et al (2008) supra).
A number of experimental animal models have addressed the role of IL-18 in cardiovascular diseases, especially the development of atherosclerosis. Administering recombinant IL-18 to ApoE−/− mice resulted in increased atherosclerotic lesion size and elevated plasma IFNγ levels (Tenger et al. (2005) Arterioscler Thromb Vasc Biol 25: 791). Overexpressing the naturally occurring IL-18 inhibitor, IL-18BP, in ApoE−/− mice resulted in reduced atherosclerosis and major changes in plaque phenotype, indicating more “stable” lesions (Mallat et al. (2001) Circ Res 89:E41). The phenotype of the double knockout ApoE−/− x IL-18−/− mice is similar to the IL-18BP overexpressing ApoE−/− mice in that the lesions are smaller and their phenotype is more “stable” (Elhage et al. (2003) Cardiovasc Res 59:234). Administering a neutralising anti-IL-18 antibody in a rat vascular injury model significantly reduced neointima formation, corresponding to reduced intra-intimal cell proliferation, and IFNγ and IL-6 gene expression (Maffia et al (2006) Circulation 114:430). Additionally, IL-18−/− mice are susceptible to hyperphagia, obesity and insulin resistance (Netea et al (2006) Nat. Med. 12:650).
As described above, IL-18 may be considered a proinflammatory cytokine which mediates disease, as well as an immunostimulatory cytokine that is important for host defense against infection and cancer.
The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural anti-inflammatory as well as immunosuppressive molecule. In humans, four different isoforms (a, b, c and d) of IL-18BP have been identified resulting from alternative splicing. Only IL-18BPa and IL-18BPc have been shown to bind and neutralise the biological activity of human IL-18, isoform a having a 10-fold higher affinity than isoform c (Kim et al (2000) PNAS 97 1190-1195). Computer modeling of human IL-18 identified two charged residues, Glu-42 and Lys-89, which interact with oppositely charged amino acid residues buried in a large hydrophobic pocket of IL-18BP. The cell surface IL-18 receptor alpha chain competes with IL-18BP for IL-18 binding, although the IL-18 receptor alpha chain does not share significant homology to IL-18BP.
The structure of soluble IL-18 has been determined by NMR spectroscopy and consists of 12 strands forming a β-trefoil (Kato et al (2003) Nat. Struct. Biol. 10:966). Three specific surface areas have been identified. Site I is formed by five residues (Arg13, Asp17, Met33, Asp 35 and Asp 132) located on one side of the core barrel and is responsible for binding to IL-18Rα. Site II is formed by six residues (Lys4, Leu5, Lys8, Arg 58, Met60 and Arg104) located at the top of the β-barrel and is also important for IL-18Rα binding. Site III (Lys 79, Lys 89, asp98) located opposite to site II at the bottom of the β-barrel is binding to IL-18Rβ (Kato et al. (2003) supra).
Antibodies that bind IL-18 are known in the art (see for example U.S. Pat. No. 6,706,487, WO 2001/058956, EP 1621616, US 2005/0147610; EP 0 974 600; and WO 0158956). However, there is still a need for anti-IL-18 antibodies that reduce the biological activity of IL-18 in a therapeutic context.